The silicone depletion in combination products induced by biologics.

Silicone oil (SO) migration into the drug product of combination products for biopharmaceuticals during storage is a common challenge. As the inner barrel surface is depleted of SO the extrusion forces can increase compromising the container functionality. In this context we investigated the impact of different formulations on the increase in gliding forces in a spray-on siliconized pre-filled syringe upon storage at 2-8 °C, 25 °C and 40 °C for up to 6 months. We tested the formulation factors such as surfactant type, pH, and ionic strength in the presence of one monoclonal antibody (mAb) as well as compared three mAbs in one formulation. After 1 month at 40 °C, the extrusion forces were significantly increased due to SO detachment dependent on the fill medium. The storage at 40 °C enhanced the SO migration process but it could also be observed at lower storage temperatures. Regarding the formulation factors the tendency for SO migration was predominantly dependent on the presence and type of surfactant. Interestingly, when varying the mAb molecules, one of the proteins showed a rather stabilizing effect on the SO layer resulting into higher container stability. In contrast to the formulation factors, those different stability outcomes could not be explained by interfacial tension (IFT) measurements at the SO interface. Further characterization of the mAb molecules regarding interfacial rheology and conformational stability were not adequately able to explain the observed difference. Solely a hydrophobicity ranking of the molecules correlated to the stability outcome. Further investigations are needed to clarify the role of the protein in the SO detachment process and to understand the cause for the stabilization. However, the study clearly demonstrated that the protein itself plays a critical role in the SO detachment process and underlined the importance to include verum for container stability.

Dynamical Transition in Dehydrated Proteins.

Terahertz time-domain spectroscopy and differential scanning calorimetry were used to study the role of the dynamics of biomolecules decoupled from solvent effects. Lyophilized sucrose exhibited steadily increasing absorption with temperature as anharmonic excitations commenced as the system emerged from a deep minimum of the potential energy landscape where harmonic vibrations dominate. The polypeptide bacitracin and two globular proteins, lysozyme and human serum albumin, showed a more complex temperature dependence. Further analysis focused on the spectral signature below and above the boson peak. We found evidence of the onset of anharmonic motions that are characteristic for partial unfolding and molecular jamming in the dry biomolecules. The activation of modes of the protein molecules at temperatures comparable to the protein dynamical transition temperature was observed in the absence of hydration. No evidence of Fröhlich coherence, postulated to facilitate biological function, was found in our experiments.

Molecular Dynamics Study of Protein Aggregation at Moving Interfaces.

Repeated compression and dilation of a protein film adsorbed to an interface lead to aggregation and entry of film fragments into the bulk. This is a major mechanism for protein aggregate formation in drug products upon mechanical stress, such as shaking or pumping. To gain a better understanding of these events, we developed a molecular dynamics (MD) setup, which would, in a later stage, allow for in silico formulation optimization. In contrast to previous approaches, the molecules of our model protein human growth hormone displayed realistic shapes, surfaces, and interactions with each other and the interface. This enabled quantitative assessment of protein cluster formation. Simulation outcomes aligned with experimental data on subvisible particles and turbidity, thereby validating the model. Computational and experimental results indicated that compression speed does not affect the aggregation behavior of preformed protein films but rather their regeneration. Protein clusters that formed during compression disassembled upon relaxation, suggesting that the particles originate from a partly compressed state. Desorption studies via steered MD revealed that proteins from compressed systems are more likely to detach as clusters, implying that compression effects at the interface translate into aggregates present in the bulk solution. With the possibility of studying the impact of different variables upon compression and dilation at the interface on a molecular level, our model contributes to the understanding of the mechanisms of protein aggregation at moving interfaces. It also enables further studies to change formulation parameters, interfaces, or proteins.

Closing the Gap between Experiment and Simulation─A Holistic Study on the Complexation of Small Interfering RNAs with Polyethylenimine.

Rational design is pivotal in the modern development of nucleic acid nanocarrier systems. With the rising prominence of polymeric materials as alternatives to lipid-based carriers, understanding their structure-function relationships becomes paramount. Here, we introduce a newly developed coarse-grained model of polyethylenimine (PEI) based on the Martini 3 force field. This model facilitates molecular dynamics simulations of true-sized PEI molecules, exemplified by molecules with molecular weights of 1.3, 5, 10, and 25 kDa, with degrees of branching between 50.0 and 61.5%. We employed this model to investigate the thermodynamics of small interfering RNA (siRNA) complexation with PEI. Our simulations underscore the pivotal role of electrostatic interactions in the complexation process. Thermodynamic analyses revealed a stronger binding affinity with increased protonation, notably in acidic (endosomal) pH, compared to neutral conditions. Furthermore, the molecular weight of PEI was found to be a critical determinant of binding dynamics: smaller PEI molecules closely enveloped the siRNA, whereas larger ones extended outward, facilitating the formation of complexes with multiple RNA molecules. Experimental validations, encompassing isothermal titration calorimetry and single-molecule fluorescence spectroscopy, aligned well with our computational predictions. Our findings not only validate the fidelity of our PEI model but also accentuate the importance of in silico data in the rational design of polymeric drug carriers. The synergy between computational predictions and experimental validations, as showcased here, signals a refined and precise approach to drug carrier design.

Three-Dimensional Homodyne Light Detection (3D-HLD) for High-Throughput Submicron Particle Analysis in (Highly Concentrated) Protein Biopharmaceuticals, Viral Vectors, and LNPs.

During biopharmaceutical development, particle monitoring and characterization are crucial. Notably, particles can be impurities considered as critical quality attribute, or active pharmaceutical ingredient (e.g., viral vectors) or drug delivery system (e.g., lipid nanoparticles) itself. Three-dimensional homodyne light detection (3D-HLD) is a novel technique that can characterize particles in the ∼0.2 µm to 2.0 µm size range. We evaluated 3D-HLD for the analysis of high concentration protein formulations (up to 200 mg/mL), where formulation refractive index and background noise became limiting factors with increasing protein concentration. Sample viscosity however did not impact 3D-HLD results, in contrast to comparative analyses with NTA and MRPS. We also applied 3D-HLD in high-throughput screenings at high protein concentration or of lipid nanoparticle and viral vector formulations, where impurities were analyzed in the presence of a small (<0.2 µm) particulate active pharmaceutical ingredient. 3D-HLD turned out to be in good agreement with or a good complement to other state-of-the-art particle characterization techniques, including BMI, MRPS, and DLS. The main application of 3D-HLD is high-throughput particle analysis at low sample volume. Follow-up investigation of the optimized particle sizing approach and of detection settings could further improve the understanding of the method and potentially increase ease of operation.

Genome length determination in adeno-associated virus vectors with mass photometry.

Recombinant adeno-associated viruses (rAAVs) are attractive therapeutic viral vectors for gene delivery. To ensure the efficacy and safety of rAAV-based therapies, comprehensive characterization of the adeno-associated virus (AAV) capsids is essential. Mass photometry (MP) provides the advantage of short analysis times, low sample consumption, and high accuracy of molecular mass determination. Despite having just recently emerged, MP has already been used to characterize AAV genome content and quantify filled/empty capsid ratios. In this study, we explored three approaches for the application of MP to assess genome length in AAVs. In approach 1, genome length in intact AAVs was approximated with good precision (coefficient of variation [%CV] < 2.6%) and accuracy (±5%) by using a straightforward protein-based calibration. In approach 2, genome length was determined even more accurately (±1%, %CV < 2.9%) considering calibration with a set of additional AAVs of different genome length. In approach 3, genome length was assessed after genome release from the capsid by heating in 1% sodium dodecyl sulfate followed by surfactant removal with precision of %CV < 0.7% and accuracy of ±5%. In conclusion, the three developed MP-based approaches are fast, precise, and accurate methods for genome length determination in AAVs, differing in their calibration materials and efforts.

Lyophilization cycle design for highly concentrated protein formulations supported by micro freeze-dryer and heat flux sensor.

High-concentration protein formulations (HCPFs) represent a common strategy and freeze-drying can mitigate the stability challenges of HCPFs. In general, an in-depth characterization of the lyophilization process is essential to not impair the product quality by inappropriate process parameters. The aim of this study was to create a primary drying design space for lyophilized HCPFs by utilizing the heat flux sensor (HFS) integrated in a MicroFD with a minimum number of cycles and product vials. All the necessary data to obtain the design space were determined starting from only two lyophilization cycles, each holding 19 vials. The vial heat transfer coefficient (Kv) was determined by the HFS and compared to gravimetric values. The results indicate a consistant offset between the HFS and the gravimetry based values for annealed samples with higher protein content. This work highlights a possibility of integrating new technologies, the HFS and the MicroFD to generate a design space for lyophilization of HCPFs, which enables to implement a QbD approach at minimal material and time investment.

Evaluation of a novel silicone oil free primary packaging system with PTFE-based barrier stopper for biologics.

In order to overcome silicone oil related problems for biopharmaceuticals, novel container systems are of interest with a focus on the reduction, fixation or complete avoidance of silicone oil in the primary container. Ultimately, silicone oil free (SOF) container systems made from cyclic olefin (co-)polymer or glass combined with the respective silicone-oil free plungers were developed. In the following study we evaluated the potential of a SOF container system based on a glass barrel in combination with a fluoropolymer coated syringe plunger. In a long-term stability study, the system was compared to other alternative container systems in terms of functionality and particle formation when filled with placebo buffers. The system proved to be a valuable alternative to marketed siliconized container systems with acceptable and consistent break-loose gliding forces and it was clearly superior in terms of particle formation over storage time. Additionally, we evaluated the importance of the glass barrel surface for functionality. The interaction of the fill medium with the glass surface significantly impacted friction forces. Consequently, storage conditions and production processes like washing and sterilization, which can easily alter the surface properties, should be carefully evaluated, and controlled. The novel combination of non-lubricated glass barrel and fluoropolymer coated plunger provides a highly valuable SOF packaging alternative for biopharmaceuticals.

Topical ocular protein delivery based on protein powder suspensions in semifluorinated alkanes.

The field of ocular diseases, specifically retinal diseases is a successful target area for protein drugs with various marketed products. Besides the intraocular treatment of the retina, the topical treatment of corneal or conjunctival diseases is a promising approach. Topical ocular protein formulations face the challenges of poor penetration and potentially low stability. In this study we tested suspensions based on the semifluorinated alkane F6H8 to improve the topical ocular protein delivery. Such suspensions are well known for the increased protein stability compared to aqueous solutions. Furthermore, F6H8 is well known as vehicle for ocular delivery due to its easy spreading on the cornea. Penetration of a model mAb and its Fab fragment was tested in an ex vivo corneal penetration test. The amount of penetrated protein was increased when the protein powder suspensions were used compared to the respective aqueous solutions. Sodium caprate as penetration enhancer at 5 mg/ml substantially increased the Fab fragment (7-fold) and the mAB (3-fold) concentration in the corneal tissue when applied as an aqueous solution. The effect was surprisingly more pronounced, when Fab fragment (31-fold) or mAb (13-fold) and the penetration enhancer were formulated as F6H8 suspensions. The same penetration enhancement from suspensions could be achieved with 2.5 mg/ml, but the penetration was reduced compared to 2.5 mg/ml in the aqueous solution. A test based on stratified human keratinocytes did not indicate eye irritation by the tested formulations. Furthermore, stability studies for bevacizumab suspensions in semifluorinated alkanes were investigated and showed superior long-term stability compared to the marketed aqueous solution. Overall results demonstrate the high potential of topical ocular protein delivery using powder suspensions in non-aqueous vehicles based on semifluorinated alkanes.

A model-based optimization strategy to achieve fast and robust freeze-drying cycles.

Freeze-drying is a time and cost-intensive process. The primary drying phase is the main target in a process optimization exercise. Biopharmaceuticals require an amorphous matrix for stabilization, which may collapse during primary drying if the critical temperature of the formulation is exceeded. The risk of product collapse should be minimized during a process optimization to accomplish a robust process, while achieving an economical process time. Mechanistic models facilitate the search for an optimal primary drying protocol. We propose a novel two-stage shelf temperature optimization approach to maximize sublimation during the primary drying phase, without risking product collapse. The approach includes experiments to obtain high-resolution variability data of process parameters such as the heat transfer coefficient, vial dimensions and dried layer resistance. These process parameters variability data are incorporated into an uncertainty analysis to estimate the risk of failure of the protocol. This optimization approach enables to identify primary drying protocols that are faster and more robust than a classical approach. The methodology was experimentally verified using two formulations which allow for either aggressive or conservative freeze-drying of biopharmaceuticals.

Chemometrics in Protein Formulation: Stability Governed by Repulsion and Protein Unfolding.

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.

Impact of autoclavation on baked-on siliconized containers for biologics.

Many pharmaceutical manufacturing units utilize pre-sterilized ready-to fill primary containers for parenterals. The containers may have been sterilized by the supplier via autoclavation. This process can change the physicochemical properties of the material and the subsequent product stability. We studied the impact of autoclavation on baked on siliconized glass containers for biopharmaceuticals. We characterized the container layers of different thickness before and after autoclavation for 15 min at 121 °C and 130 °C. Furthermore, we analyzed the adsorption of a mAb to the silicone layer and subjected filled containers to 12 weeks storage at 40 °C monitoring functionality and subvisible particle formation of the product. Autoclavation turned the initially homogenous silicone coating into an incoherent surface with uneven microstructure, changed surface roughness and energy, and increased protein adsorption. The effect was more pronounced at higher sterilization temperatures. We did not observe an effect of autoclavation on stability. Our results did not indicate any concerns for autoclavation at 121 °C for safety and stability of drug/device combination products using baked-on siliconized glass containers.

Afraid of the wall of death? Considerations on monoclonal antibody characteristics that trigger aggregation during peristaltic pumping.

Protein aggregation is of major concern in manufacturing of biopharmaceutics. Protein aggregation upon peristaltic pumping for filtration, transfer or filling is triggered by protein adsorption to the tubing surface and subsequent film rupture during roller movement. While the impact of tubing type and formulation has been studied in more detail, the contribution of the protein characteristics is not fully resolved. We studied the aggregation propensity of six monoclonal antibodies during peristaltic pumping and characterized their colloidal and conformational stability, hydrophobicity, and surface activity. A high affinity to the surface resulting in faster adsorption and film renewal was key for the formation of protein particles ≥ 1 µm. Film formation and renewal were influenced by the antibody hydrophobicity, potential for electrostatic self-interaction and conformational stability. The initial interfacial pressure increase within the first minute can serve as a good predictor for antibody adsorption and particle formation propensity. Our results highlight the complexity of protein adsorption and emphasize the importance of formulation development to reduce protein particle formation by avoidance of adsorption to interfaces.

SV-AUC as a stability-indicating method for the characterization of mRNA-LNPs.

During the SARS-CoV2 pandemic mRNA vaccines in the form of lipid nanoparticles (LNPs) containing the mRNA, have set the stage for a new area of vaccines. Analytical methods to quantify changes in size and structure of LNPs are crucial, as changes in these parameters could have implications for potency. We investigated the application of sedimentation velocity analytical ultracentrifugation (SV-AUC) as quantitative stability-indicating method to detect structural changes of mRNA-LNP vaccines upon relevant stress factors (freeze/thaw, heat and mechanical stress), in comparison to qualitative dynamic light scattering (DLS) analysis. DLS was capable to qualitatively determine size and homogeneity of mRNA-LNPs with sufficient precision. Stress factors, in particular freeze/thaw and mechanical stress, led to increased particle size and content of larger species in DLS and SV-AUC. Changes upon heat stress at 50 °C were only detected as increased flotation rates by SV-AUC. In addition, SV-AUC was able to observe changes in particle density, which cannot be detected by DLS. In conclusion, SV-AUC can be used as a highly valuable quantitative stability-indicating method for characterization of LNPs.

Drug Product Characterization of High Concentration Non-Aqueous Protein Powder Suspensions.

High concentration protein formulations for subcutaneous injection represent a substantial number of development projects in the pharmaceutical industry. Such concentrated aqueous protein solutions face some specific challenges such as increased viscosity and aggregation propensity. Protein powder suspensions in non-aqueous vehicles could be an alternative providing lower viscosity than the respective aqueous solution. The choice of potential suspension vehicles is limited as traditional non-aqueous liquids, such as oils, show an inherent high viscosity. We studied suspensions prepared by dispersing spray-dried protein powder in different vehicles including sesame oil and medium chain triglycerides, as well as fluorinated and semifluorinated alkanes. We found, that semifluorinated alkanes enable formulations with high concentrations up to 280 mg/ml monoclonal antibody with a low viscosity of less than 10 mPa·s and low injection forces. The glide force of suspensions containing 210 mg/ml protein was not affected by the particle size of the spray-dried powders with medians ranging from 1 to 14 µm. In contrast, suspensions prepared with cryo-milled powder showed markedly higher viscosities and were not injectable at the same concentration. Protein powder suspensions were syringeable using a 25G needle. Vial filling using a peristaltic pump was possible and lead to a uniform filling. Sedimentation of the suspension was slow and does not lead to challenges upon vial filling during manufacturing or transfer of the suspension into syringes. Thus, we could show that dispersions of spray-dried protein powders in non-aqueous vehicles, such as semifluorinated alkanes, are a promising alternative to aqueous protein solutions at high concentrations.

Effect of the Tubing Material Used in Peristaltic Pumping in Tangential Flow Filtration Processes of Biopharmaceutics on Particle Formation and Flux.

Tangential flow filtration (TFF) is a central step in manufacturing of biopharmaceutics. Membrane clogging leads to decreased permeate flux, longer process time and potentially complete failure of the process. The effect of peristaltic pumping with tubings made of three different materials on protein particle formation during TFF was monitored via micro flow imaging, turbidity and photo documentation. At low protein concentrations, pumping with a membrane pump resulted in a stable flux with low protein particle concentration. Using a peristaltic pump led to markedly higher protein particle formation dependent on tubing type. With increasing protein particle formation propensity of the tubing, the permeate flux rate became lower and the process took longer. The protein particles formed in the pump were captured in the cassette and accumulated on the membrane leading to blocking. Using tubing with a hydrophilic copolymer modification counteracted membrane clogging and flux decrease by reducing protein particle formation. In ultrafiltration mode the permeate flux decrease was governed by the viscosity increase rather than by the protein aggregation; but using modified tubing is still beneficial due to a lower particle burden of the product. In summary, using tubing material for peristaltic pumping in TFF processes which leads a less protein particle formation, especially tubing material with hydrophilic modification, is highly beneficial for membrane flux and particle burden of the product.

Modification of Tubings for Peristaltic Pumping of Biopharmaceutics.

Protein particle formation during peristaltic pumping of biopharmaceuticals is due to protein film formation on the inner tubing surface followed by rupture of the film by the roller movement. Protein adsorption can be prevented by addition of surfactants as well as by increasing the hydrophilicity of the inner surface. Attempts based on covalent surface coating were mechanically not stable against the stress of roller movement. We successfully incorporated surface segregating smart polymers based on a polydimethylsiloxane (PDMS) backbone and polyethylene glycol (PEG) side blocks in the tubing wall matrix. For this we applied an easy, reproducible and cost-effective process based on soaking of tubing in toluene containing the PDMS-PEG copolymer. With this tubing modification we could drastically reduce protein particle formation during peristaltic pumping of a monoclonal antibody and human growth hormone (HGH) formulation in silicone and thermoplastic elastomer-based tubing. The modification did not impact the tubing integrity during pumping while hydrophilicity was increased and protein adsorption was prevented. Free PDMS-PEG copolymer might have an additional stabilizing effect, but less than 50 ppm of the PDMS-PEG copolymer leached from the modified tubing during 1 h of pumping in the experimental setup. In summary, we present a new method for the modification of tubings which reduces protein adsorption and particle formation during any operation involving peristaltic pumping, e.g. transfer, filling, or tangential flow filtration.

Reaching the breaking point: Effect of tubing characteristics on protein particle formation during peristaltic pumping.

Peristaltic pumping has been identified as a cause for protein particle formation during manufacturing of biopharmaceuticals. To give advice on tubing selection, we evaluated the physicochemical parameters and the propensity for tubing and protein particle formation using a monoclonal antibody (mAb) for five different tubings. After pumping, particle levels originating from tubing and protein differed substantially between the tubing types. An overall low shedding of tubing particles by wear was linked to low surface roughness and high abrasion resistance. The formation of mAb particles upon pumping was dependent on the tubing hardness and surface chemistry. Defined stretching of tubing filled with mAb solution revealed that aggregation increased with higher strain beyond the breaking point of the protein film adsorbed to the tubing wall. This is in line with the decrease in protein particle concentration with increasing tubing hardness. Furthermore, material composition influenced particle formation propensity. Faster adsorption to materials with higher hydrophobicity is suspected to lead to a higher protein film renewal rate resulting in higher protein particle counts. Overall, silicone tubing with high hardness led to least protein particles during peristaltic pumping. Results from this study emphasize the need of proper tubing selection to minimize protein particle generation upon pumping.

Evaluation of adsorption of DNA/PEI polyplexes to tubing materials.

Nucleic acid drugs hold great promise for potential treatment of a variety of diseases. But efficient delivery is still the major challenge impeding translation. Nanoformulations based on polymers and lipids require preparation processes such as microfluidic mixing, spray drying or final filling, where pumping is a crucial step. Here, we studied the effect of pumping on the component and overall loss of a binary polyplex formulation made of DNA and polyethyleneimine (PEI). We varied tubing length and material with a focus on subsequent spray drying. Interestingly, product loss increased with the length of silicon tubing. Losses of DNA were prevented by using Pumpsil. The following spray drying process did not affect DNA content but caused PEI loss. Characterization of the different tubing materials revealed similar hydrophobicity of all tubing materials and showed neutral Pumpsil® surface charge, negative Santoprene™ surface charge, and a positive Silicon surface charge. Hence, adsorption of DNA onto tubing material was concluded to be the root cause for DNA loss after pumping and is based upon an interplay of ionic and hydrophobic interactions between polyplexes and tubing material. Overall, selecting the appropriate tubing material for processing nucleic acid nanoparticles is key to achieving satisfactory product quality.

Utility of Three Flow Imaging Microscopy Instruments for Image Analysis in Evaluating four Types of Subvisible Particle in Biopharmaceuticals.

Subvisible particles (SVPs) are a critical quality attribute of parenteral and ophthalmic products. United States Pharmacopeia recommends the characterizations of SVPs which are classified into intrinsic, extrinsic, and inherent particles. Flow imaging microscopy (FIM) is useful as an orthogonal method in both the quantification and classification of SVPs because FIM instruments provide particle images. In addition to the conventionally used FlowCam (Yokogawa Fluid Imaging Technologies) and Micro-Flow Imaging (Bio-Techne) instruments, the iSpect DIA-10 (Shimadzu) instrument has recently been released. The three instruments have similar detection principles but different optical settings and image processing, which may lead to different results of the quantification and classification of SVPs based on the information from particle images. The present study compares four types of SVP (protein aggregates, silicone oil droplets, and surrogates for solid free-fatty-acid particles, milled-lipid particles, and sprayed-lipid particles) to compare the results of size distributions and classification abilities obtained using morphological features and a deep-learning approach. Although the three FIM instruments were effective in classifying the four types of SVP through convolutional neural network analysis, there was no agreement on the size distribution for the same protein aggregate solution, suggesting that using the classifiers of the FIM instruments could result in different evaluations of SVPs in the field of biopharmaceuticals.